Human mitoNEET is a homodimeric iron-sulfur protein located in the outer mitochondrial membrane with unknown function, but which is known to interact with thiazolidinedione diabetes drugs Each monomer houses a [2Fe-2S] cluster with an unusual (Cys)(3)(His)(1) ligation The His ligand is important for enabling cluster release and for tuning the redox potential We use multifrequency (X-, Ka-, and Q-band) and multitechnique (continuous-wave, electron spin-echo envelope modulation (ESEEM), pulsed electron-nuclear double resonance (ENDOR), and hyperfine sublevel correlation (HYSCORE)) electron paramagnetic resonance spectroscopy to investigate the cluster in its paramagnetic reduced [Fe2+Fe3+] (S = 1/2) state. It has a rhombic g tensor (2.007, 1 937, 1 897) with an average g value of 1 947 that falls between those of Rieske-type and ferredoxin-type [2Fe-2S] clusters Simulation and least-squares fitting of orientation-selective Ka- and Q-band ENDOR, 1 D ESEEM, and HYSCORE spectra of N-14 and N-15-labeled mitoNEET yield the principal values and orientations of both the hyperfine tensor (N-14, A(ISO) = -6 25 MHz, T = -0 94 MHz) and the quadrupolar tensor (e(2)Qq/h = -2 47 MHz, eta = 0 38) of the ligating histidine nitrogen N-delta. From these, we can infer the absolute g tensor orientation with respect to the cluster: The g(2) axis is close to perpendicular to the [2Fe-2S] plane, and g(1) and g(3) are in-plane, but skewed from the Fe-Fe and S-S axes. In X-band ENDOR and ESEEM spectra, a weakly coupled nitrogen is visible, most likely the N, of the histidine in the protonated state We find that the cluster is in a valence-localized state, where Fe2+ is His-bound The field-sweep spectra show evidence of intercluster dipolar coupling that can be simulated using an uncoupled spin model for each cluster (SFe2+ = 2, SFe3+ = 5/2). The parameters determined in this work can function as reporters on how the cluster structure is altered upon pH changes and drug binding